GLUTAMATE RECEPTOR (OR EAA RECEPTOR) POLYNUCLEOTIDES AND THEIR USES

Abstract: Human CNS receptor-encoding DNA is provided from which more than one form of the receptor protein, an edited form and an unedited form, is expressed <u>in vivo</u>. The edited and unedited receptor forms are functionally distinct, and appear to be selectively expressed. Thus, a CNS receptor-encoding gene that is subject to such editing has value in screening compounds for potential therapeutic utility.

Description: GLUTAMATE RECEPTOR (OR EAA RECEPTOR) POLYNUCLEOTIDES AND THEIR USES Field of the Invention This invention relates to novel CNS receptor polynucleotides and the proteins they encode, and their use in screening potential therapeutic compounds. Background to the Invention In the mammalian central nervous system (CNS) , the transmission of nerve impulses is controlled by the interaction between a neurotransmitter substance released by the "sending" neuron and a surface receptor on the "receiving" neuron to which the neurotransmitter binds causing excitation thereof . There are a number of neurotransmitters in the CNS, each of which target specific receiving neurons. For example, glutamate, dopamine and serotonin neurotransmitters each target a different family of receptors. Glutamate, which is referred to as an excitatory amino acid (EAA) , interacts with receptors variously referred to as glutamate or EAA receptors, while dopamine and serotonin interact specifically with dopamine and serotonin receptors, respectively. Within each receptor family, the receptors are classified by their ligand-binding or functional characteristics. For example, some EAA receptors are classified according to their differential binding to the agonists, NMDA (N-methyl-D-aspartate) , AMPA ( alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate) , and kainate (2-carboxy-4- (1-methylethenyl) -3-pyrrolidineacetate) . Thus, NMDA receptors bind glutamate and bind NMDA with greater affinity than kainate or AMPA, while AMPA and kainate receptors bind glutamate, and bind AMPA and kainate, respectively, with greater affinity than other agonists . In contrast to dopamine and serotonin receptors, some EAA receptors are functional in an electrophysiological sense as SUBSTITUTESHEET determined by established electrophysiological assays such as that described by Hollman et al. in Nature 342: 643, 1989, or by any other assay appropriate for detecting conductance across a cell membrane. In essence, EAA receptors form ligand-gated ion channels. Thus, in response to binding an appropriate ligand, e.g., glutamate, AMPA, kainate or NMDA, an EAA receptor ion channel will "open" or become more permeable to allow the influx of cation that is required for normal synaptic transmission. In the absence of ligand binding, the ion channels remain "closed" or less permeable to cation, preventing the inward flow of cation required for synaptic transmission. At least six AMPA-type rodent receptors have been cloned, and named GluR-1 to 6. Expression studies suggest that GluR-2 is the dominant subunit in determining functional properties associated with Ca 2+ permeability in this rodent receptor family. Mutation studies have shown that this permeability is determined by a single amino acid, arginine (R) , in the putative channel-forming transmembrane II (TMII) of rat GluR-2; a glutamine (Q) residue is present in the other AMPA receptors. It was subsequently revealed that the R form of the GluR-2 receptor is generated from the same gene as the Q form by an RNA editing process, indicating that, in rat brain, the occurrence of this "editing" process determines cation flow in GluR-2 channels (Sommer et al, 1991, Cell, 67:11) . Reports to date have found almost 100% efficiency of the editing process for rodent GluR-2 with low level expression of unaltered Q forms in the developing central nervous system (Sommer et al, supra; and see Burnashev et al, 1992, Neuron, 8:189) . Most recently, the AMPA-type rat receptors GluR- 5 and GluR-6 have also been shown to undergo RNA editing (Sommer et al, supra; Burnashev et al, supra; and see Kohler et al, 1993, Neuron, 10:491) . RNA editing is a relatively rare phenonmenon, but occurs in various organisms and may involve a number of different mechanisms. The editing of the rodent AMPA receptor, GluR-2, has been demonstrated to require a base paired intron/exon structure. SUBSTITUTE SH ET A nuclear adenosine deaminase specific for double-stranded DNA is postulated to be involved in the base conversion, although direct evidence of the mechanism and any regulation of the process remain to be investigated. Several human glutamate receptors have been cloned, including those of the AMPA-type such as hGluRl (Puckett et al, 1991, Proc. Natl. Acad. Sci., 88:7557), hGluR-2, hGluR-3 (Biochem. Biophys. Acta, 1994, 1219:563) and those of the kainate-type, such as humEAAl (EP 529,994); humEAA2 (EP 529,995); humEAA3 (EP 617,123) and humEAA4 (EP 578,409) . The human glutamate receptors are of great medical importance because of their postulated role in the mediation of learning and memory acquisition. In addition, excitatory amino acids can be highly toxic to neurons and dysfunction of this neurotransmitter system has been implicated in several neurological disorders such as Alzheimer's disease, Huntington's chorea, epilepsy, Parkinson's disease, amyotrophic lateral sclerosis, AIDS encephalopathy and dementia complex. To date, the RNA editing phenomenon, an important determinate of the functional properties of CNS receptors and particularly glutamate receptors, has not been observed in humans. Summary of the Invention It has now been discovered that the synthesis of human CNS receptors in vivo is regulated by an editing mechanism. This "editing" results in the expression from a single human CNS receptor gene of structurally distinct forms of the encoded CNS receptor protein, i.e. edited and unedited receptor forms. It is postulated that certain neurodegenerative disease states are associated with an aberrant editing mechanism. The evidence presented here further indicates that this editing mechanism is operative in a tissue-selective manner, and in a developmentally regulated manner. Thus, the expression products of a given CNS receptor-encoding gene are valuable in screening compounds for potential therapeutic utility, and particularly in selecting drug UBSTITUT candidates that interact selectively with edited human CNS receptor forms. Accordingly, the invention provides, in one of its aspects, a method for identifying a human CNS receptor-selective ligand, which comprises: a) determining the interaction between a candidate ligand and a first human CNS receptor of a type that is subject to editing; b) determining the interaction between the candidate ligand and a second human CNS receptor which receptor is an editing-altered variant of said first receptor; and either c) selecting that candidate ligand which interacts selectively with one of said receptors, or d) selecting that candidate ligand which interacts substantially equivalently with both of said receptors. This method of the present invention is performed, in embodiments of the present invention, using first and second human glutamate receptors, and particularly using human glutamate receptors of the AMPA-type or kainate-type. For use in the method of the present invention, the invention further provides cells that are transformed to express novel forms of edited and unedited human CNS receptors, and particularly edited and unedited forms of human glutamate receptors. For use in constructing such cells, the invention provides, in a related aspect, isolated polynucleotides that encode such receptors. In a further aspect, the invention provides a method useful to identify agents that modulate the editing of human CNS receptors in vivo, which comprises: SUBSTITUTESHEET a) obtaining a human neuronal cell line that (1) incorporates DNA coding for the unedited form of an edited human CNS receptor, and that (2) elaborates, upon culturing, the edited form of the receptor; b) culturing the cell line in the presence of a candidate modulator of said editing; and c) determining the effect of said modulator on the elaboration of said edited form of said receptor. These and other aspects of the invention are described in greater detail with reference to the accompanying drawings, in which: Brief Reference to the Drawings Figure 1 provides the genomic DNA and protein sequences of the unedited form of the human GluR2B receptor; Figure 2 is a comparison of the partial amino acid sequences of the edited and unedited forms of the GluR2B receptor,- Figure 3 illustrates the exon of the GluR2B gene which is subject to editing, and the primers used in genomic DNA isolation; and Figure 4 illustrates the results of an enzymatic digestion of human genomic GluR2B DNA. Figure 5 provides the genomic DNA and protein sequences of the unedited form of the human EAA3 receptor; Figure 6 provides the genomic DNA and protein sequences of the unedited form of human EAA4 receptor; Figure 7 illustrates editing in human EAA3 and EAA4 receptors: a) comparison of genomic and cDNA nucleotide sequences and amino acid sequences in TMI; b) comparison of genomic and cDNA nucleotide sequences and amino acid sequences in TMII; and Detailed Description of the Invention and Its Preferred Embodiments The invention is based on the present discovery that a single TIT T gene coding for a human CNS receptor, when expressed in vivo, can yield not only a receptor of the amino acid sequence determined by that gene, but can also yield one or more forms of the receptor that are not encoded by that gene. This editing phenomenon was revealed by comparing the cDNA sequence of a given human receptor with the counterpart genomic DNA sequence for the given receptor. Sequence discrepancies revealed that the cDNA sequence had been altered relative to the genomic sequence, with the result that the cDNA encoded a receptor protein having at least one amino acid substitution relative to the receptor encoded by the genomic DNA. In this way, the editing phenomenon yields receptor forms that differ in terms of receptor protein structure, and in some instances also in terms of receptor protein function. These different receptor forms are the expression products of polynucleotides characterized herein as "edited" and "unedited" . "Unedited" polynucleotides are those having a genomically encoded sequence. Similarly, in "unedited" receptor proteins, each amino acid in the receptor protein sequence has an appropriate source codon within the genomic DNA sequence, i.e., the unedited polynucleotide, from which it is expressed. "Edited" receptor proteins, on the other hand, are expressed from unedited genomic polynucleotides, yet have a receptor protein sequence in which at least one amino acid is not represented in the unedited polynucleotide from which it is expressed. The terms "edited" and "unedited" are also used herein with respect to mRNA, sDNA and cDNA sequences of the respective receptor proteins. The term "distinct", as it is used herein with respect to edited and unedited CNS receptors, refers to the differences between the edited and unedited receptors, which include at least one of a structural difference, i.e. an amino acid sequence difference, or a functional difference, i.e. a difference in ligand binding or electrophysiological properties which can be determined using assays appropriate for determining ligand/receptor interaction. The term "functionally distinct" indicates that each of the BSTITUTESH ET edited and unedited receptor forms reacts differently to a given stimulus. For example, functionally distinct forms of an EAA receptor may be represented by an unedited receptor form which exhibits ligand-gated ion channel activity in response to a given ligand, while the edited form of the receptor does not exhibit channel activity in the presence of that ligand. Functionally distinct forms of a CNS receptor may also have distinct ligand- binding properties. While not being limited to any single theory with regard to the mechanism of editing, it is believed that the "editing" of a gene is catalyzed by an enzyme at the level of transcription. Thus, the "editing" enzyme, during the transcription of a genomic CNS receptor-encoding DNA, recognizes a nucleotide within the DNA sequence and, instead of incorporating the appropriate corresponding nucleotide into the mRNA, it incorporates a different nucleotide into the mRNA. The editing of CNS receptor polynucleotides does not occur 100% of the time, and thus, it appears that certain conditions or signals dictate when a CNS polynucleotide will be edited and when editing will not occur. As will be described in more detail below, the editing of human CNS genes has also been found to occur tissue specifically, occurring with greater frequency in certain CNS tissues than in others. The term "genomic polynucleotide" is used herein to refer to a polynucleotide having a nucleotide sequence that corresponds with the coding sequence of the genomic DNA. Thus, a genomic polynucleotide according to the present invention may be genomic DNA, or synthetic or cDNA comprising the exonic coding sequences of the genomic DNA but lacking the non-coding intronic sequences. A genomic polynucleotide may also be RNA which corresponds to the genomic DNA sequence, i.e. in unedited form. The term "isolated" is used herein with reference to intact polynucleotides to denote polynucleotides, including both DNA and RNA, which are free from polynucleotides encoding other human TITUTESHEET proteins. With reference to a human CNS receptor protein, the term "isolated" similarly refers to a receptor protein which is free from other human proteins. Thus, in accordance with one of its aspects, the invention provides a method for identifying a ligand that interacts with the edited and/or unedited forms of a receptor, which comprises determining the interaction between the ligand candidate and a first human CNS receptor that is subject to editing, and between the ligand candidate and a second human CNS receptor that is an editing-altered variant of the first receptor, and then selecting the ligand that interacts selectively with one of said receptor forms, in the case where targetting of a drug to a particular receptor type is desired, or selecting the ligand that interacts with both receptor forms in the case where a drug that acts non- discriminately at the receptor family is desired. For use in such a screening method, it will be necessary to identify and obtain polynucleotides that encode editing altered receptor forms within a receptor gene family. To determine if a given CNS receptor-encoding gene is subject to editing, the genomic DNA sequence of the receptor should be compared to the nucleic acid and amino acid sequences derived therefrom in vivo, specifically, the mRNA sequence transcribed in vivo from the gene sequence, or its cDNA equivalent, and the protein sequence expressed therefrom. It is important to compare the gene sequence with sequences of the mRNA and protein which are derived from in vivo processing in order to detect editing of the gene. Comparison of the gene sequence with an mRNA or protein sequence which has been artificially produced, i.e. under in vitro conditions, will likely not reflect an edited sequence since editing conditions, for example, the presence of required editing enzymes, are probably not present in vitro unless specifically added. Whether knowledge of the editing of a known gene or a novel gene is sought, the general procedure for obtaining the gene and its SUBSTITUTESHEET cDNA equivalent is the same. For example, procedures such as those described by Sun et al. , in Proc. Natl. Acad. Sci. USA, 1992, 89:1443, can be used to isolate desired receptor-encoding cDNAs. Typically, in a first step, the desired cDNA sequence is obtained from a human brain cDNA library. For this purpose, it will be necessary to design and then prepare suitable nucleic acid probes with which to isolate cDNA encoding all or part of a CNS receptor. If a novel CNS receptor gene is being sought, the probes can be based on regions of CNS receptors which are believed to be conserved among certain CNS receptor types, for example, among CNS receptors of the kainate type. If a known gene is being sought, the probes used will desirably be complementary to a region of the gene which is unique to that gene. Alternatively, the cDNA sequence, if known, can be prepared using synthetic techniques by preparing and then ligating smaller fragments of the full-length cDNA. Isolation of cDNA which hybridizes to the probes is carried out in a manner well known to those in the art. Although it is possible, full- length receptor cDNAs are not generally isolated using the probes, but rather fragments of the full-length cDNAs are isolated. The full-length cDNAs are prepared by ligation of cDNA fragments, ligation sites being determined by overlapping regions of the cDNA fragments. On preparing a full-length cDNA, it can be sequenced using techniques well-known in the art such as the Sanger sequencing method. The mRNA sequence of the gene will of course correspond to the full-length cDNA. The sequence of the protein expressed therefrom can also be derived from the isolated cDNA sequence. Having prepared a full-length cDNA, it can be used, in whole or in part, as a probe to isolate the desired corresponding genomic receptor-encoding DNA from a genomic DNA library. Again, this exercise will likely not render a full-length genomic DNA; however, isolated fragments of the genomic DNA can be used to prepare a full-length genomic receptor-encoding DNA. In order to facilitate isolation of a particular genomic DNA, the desired DNA can first be amplified to increase its quantity in relation SUBSTIT to the rest of the DNA in the library. This is typically accomplished using the well-established PCR technique. In this case, PCR primers, which are short DNA fragments known to hybridize to the terminal ends of the desired genomic DNA are added to the library and under appropriate PCR conditions, the desired DNA is amplified, thereby facilitating its detection and isolation. Once isolated and/or prepared into its full-length form, the gene can be sequenced, and its sequence can then be compared to that of the isolated cDNA. Any discrepancies between the sequences indicate the occurrence of in vivo sequence editing. It will be appreciated by those of skill in the art that it may not be necessary to sequence the entire cDNA and genomic DNA polynucleotides in order to determine, or postulate, that editing of the particular gene may have occurred. In one alternative method, only a portion of each of the cDNA and gene is sequenced in the search for sequence discrepancies, or editing. In this regard, it will be necessary to chose a region of the gene in which editing is likely to occur, for example, a region which encodes a functional domain of the receptor protein. The functional receptor domains will of course vary from receptor to receptor. In EAA receptor genes, the regions encoding intracellular transmembrane domains, which are important for ion channel activity, are regions which can be examined for evidence of editing. In the case of dopamine and serotonin receptors, which do not form ion channels, regions of the receptor genes which are believed to encode a ligand binding domain should can be examined for sequence discrepancies. If no editing is found in selected regions of the genomic and cDNA polynucleotides, it will then, of course, be necessary to compare the entire sequences of each in order to conclude that the particular gene is either edited or not. Another method of identifying if editing has, or may have, occurred is to compare the properties of the receptor proteins expressed from each of the isolated CNS receptor cDNA and gene. SUBSTITUTE SHEET For this purpose, the cDNA and genomic DNA are cloned into expression vectors which are used to transform suitable cells, as described in detail below, and the receptor proteins or membrane preparations produced therefrom, are isolated for comparison using the ligand-binding and/or electrophysiological assays also described in detail below. Functional differences between the proteins is also an indicator that editing of that particular CNS receptor protein occurs at some stage prior to its expression. On postulating that editing of a CNS receptor gene occurs, it will then be necessary to exclude the possibility that multiple genes or alternate exons are responsible for sequence differences between the genomic DNA and protein product, i.e. to confirm that such sequence differences evidence the occurrence of editing. At the outset, genomic DNA encoding the protein must be isolated, using specific DNA probes as described above, from genomic DNA libraries of different regions of the brain. This will ensure that if multiple genes exist, one or more of which are region- specific, their existence will not be overlooked. Further, the method of primer-based PCR amplification can be applied to amplify DNA that may be present in the DNA libraries in only very minute quantities. On isolation of genomic DNA, one way to confirm that multiple genes, i.e. a gene coding for the unedited form of the protein and a gene coding for the edited form of the protein, do not exist, or that alternatively spliced exons do not account for the different protein forms, is to identify characteristics that would exist in one form of the gene (or coding exon) but would be lacking in the other form of the gene (or coding exon) , for example, restriction enzyme sites. Thus, exposing isolates of the genomic DNA to restriction enzymes for which a recognition site is present in the region of concern will yield two fragments on analysis using probe-specific hybridization. On the other hand, exposing genomic DNA isolates to restriction enzymes for which there is no recognition site in the region of concern will SUBSTITUTESHEET yield a single fragment only on analysis using probe-specific hybridization. Inconsistencies from the expected results indicates that a multiple gene or exon may be involved, and the genomic DNA yielding the unexpected results must be fully sequenced in order to identify the reason for the inconsistency. Information about the characteristics of both the edited and unedited forms of a receptor protein would, of course, only be available if the protein sequences of each of the protein forms were known. In most cases, however, the only information that will be available is a receptor gene sequence and a protein sequence that may or may not correspond to the known gene sequence. Thus, different methods must be used to determine if RNA editing of the gene occurs, or if multiple genes or exons are involved to result in a protein that does not correspond in sequence to the gene believed to encode it. One such method is based on the fact that intronic gene sequences vary from gene to gene despite the fact that the coding (exonic) sequences of two genes may vary only slightly, for example by a single codon. In this method, isolated genomic DNA is subjected to restriction enzyme digestion and is then immobilized on a nitrocellulose filter. A labelled DNA probe directed to the target region, i.e. the region of the gene:protein discrepancy, is used to identify the enzyme-digested DNA fragments. Because the sequence of the genomic DNA is known, the existence of restriction sites in the targeted region and thus, the number of restriction fragments expected from a given enzyme digestion, will be known, and the result of more or less fragments than expected will indicate the existence of multiple genes or alternate codons. Alternatively, isolated genomic DNA which has been PCR-amplified can be fully sequenced in the search for multiple genes or alternate exons. This method is preferably used to confirm results obtained in other methods, such as those described above, or when such methods do not yield confirmative results. Full sequencing of any isolated genes or gene fragments will confirm their identity. If a gene encoding the "edited" protein is not SUBSTITUTE SHEET found on sequencing a substantial number of positive clones from each region of the brain, i.e. 50-100, then the existence of multiple genes and the existence of alternate exons can be excluded as possibilities for the protein sequence discrepancy. Further, in order to exclude the possibility that the difference between the DNA and protein sequences is not the result of random mutation, i.e. a point mutation or other form of mutation, it is important to determine that it occurs with a frequency greater than that which would be associated with random mutation. In this regard, expression of the edited and unedited forms of the protein with a frequency of greater than 1 in 1000, or 1 in 10,000, would eliminate the concern that either protein was the result of mutation, particularly since mutations in the human CNS are extremely rare. In a specific embodiment of the present invention, a human EAA receptor, namely the GluR2B receptor, which is described in co- pending United States application serial no. 07/896,937, was determined to be subject to editing as described in detail in the specific examples. Briefly, genomic DNA fragments of the GluR2B receptor, and full-length cDNA derived from the amino acid sequence of the receptor protein, were used to probe a genomic DNA library. Full-length genomic DNA, isolated using these probes, was sequenced and its sequence was then compared to the full-length GluR2B cDNA and protein sequences. A comparison of the sequences identified a single codon difference in the coding region of the transmembrane II domain. Specifically, the genomic DNA was found to encode a glutamine at position 587 of the protein sequence, while the cDNA encoded an arginine at position 587. The genomic DNA sequence coding for the GluR2B receptor is illustrated in Figure 1, and is different from the cDNA sequence illustrated in USSN 07/896,937 by the single nucleotide change of G to A at position 2134. The change is also reflected in the protein sequence of the GluR2B receptor at position 587. The change in protein sequence is illustrated in Figure 2 for greater clarity. Thus, according to one embodiment of the invention, SUBSTITUTESHEET there is provided a polynucleotide that codes for the Q-587 form of the human GluR2 receptor, particularly of the human GluR2B receptor. There is further provided a transformed cell having incorporated expressibly therein a polynucleotide encoding the Q-587 form of the human GluR2 receptor. Also provided is the Q- 587 form of the human GluR2 receptor, per se. To confirm that multiple GluR2 receptor-encoding genes do not exist, i.e. one gene encoding the unedited Q-587 form of GluR2B and another gene encoding the R-587 form of GluR2B, or that multiple exons coding for these edited and unedited forms do not exist, genomic GluR2B DNA was subjected to restriction enzyme digestion. Specifically, a genomic DNA sample was digested with a restriction enzyme (Bglll) known to have a recognition site in the exon containing the codon that is subject to "editing", while other genomic DNA samples were digested with restriction enzymes (EcoRI, Hindlll and Pstl) which did not have recognition sites in the edited exon. Following enzyme digestion, the DNA and its fragments were resolved using gel electrophoresis, and the fragments containing the "edited" exon were identified using a labelled probe specific for the exon. As was expected, two DNA fragments resulted from the Bglll digestion. The appearance of three or more bands following a Bglll enzyme digestion would have indicated either the presence of two genes in which the intronic sequences differed, or two exons both including the Bglll restriction site but having different sequences. A single band resulted in each of the EcoRI, Hindlll and Pstl DNA digestions. Again, the appearance of more than one band in these cases would have indicated either the presence of two genes or exons as described above. Finally, to confirm that the sequence difference between the genomic GluR2B DNA and the GluR2B receptor was not the result of random mutation, several GluR2B genomic and cDNA clones were sequenced to determine the frequency of the sequence change. For this purpose, various types of brain tissue were probed, as described above. The results clearly indicated that mutation was SUBSTITUTESHEET not the cause of the sequence changes that occurred between GluR2B DNA and the expressed GluR2B protein. The "editing" of GluR2B occurred with different frequencies in different tissues, for example GluR2B of the hippocampus, cerebellum and temporal cortex was 100% edited (i.e. contained the arginine at position 587) , while GluR2B of the substantia nigra was 71% edited, GluR2B of the corpus striatum was 89% edited and GluR2B of fetal brain tissue was 96% edited. In other embodiments of the present invention, human EAA3 and EAA4 receptors of the kainate family, described in EP 617,123 and EP 578,409 respectively, incorporated herein by reference, have been determined to be subject to editing. Using similar techniques that were used to determine editing in the GluR2B receptor, human EAA3 and EAA4 genomic DNA were observed to encode receptors having different amino acid sequences than their corresponding cDNA. Specifically, EAA3 genomic DNA encodes a glutamine (Q) residue at position 591 of the mature receptor protein while cDNA derived from various regions of the brain has been observed to encode arginine (R) at position 591. On the other hand, EAA4 genomic DNA was found to be edited at three sites; isoleucine at position 532 of the mature protein replaced with valine, tyrosine at position 536 replaced with cysteine and glutamine at position 586 replaced with arginine. The genomic sequences of EAA3 and EAA4 are illustrated in Figure 5 and Figure 6 respectively. The changes in protein sequence at each of these sites are the result of a single nucleotide substitution, adenosine→guanosine (A→G) as illustrated in Figure 7. Thus, in embodiments of the invention, there are provided the following human CNS receptor proteins, as well as polynucleotides encoding them, and transformed cells that expressibly incorporate such polynucleotides: (1) the R-591 and Q-591 forms of the human EAA3 receptor; (2) a form of the human EAA4 receptor protein having a novel combination of one or more of the following amino acid identifiers: Ile-532, Val-532, Tyr-536, Cys-536, Gin-586 and Arg-586. SUBSTITUTESHEET The glutamine to arginine (Q/R) replacement in both EAA3 and EAA4 occur in the transmembrane II (TMII) region of the receptor protein. The additional I/V 532 and Y/C 536 editing sites in EAA4 occur in the TMI region adding further complexity by enabling up to eight isoforms of EAA4 to be generated. Hence RNA editing of EAA4 results in a mosaic of receptors which may regulate glutamate activated Ca 2+ influx in the brain. The frequency of edited codons was examined in tissue from various regions of the brain and found to be differentially represented. Of the eight possible isoforms, five were observed in the various regions; specifically I.C.R, V.C.R, I.Y.Q, V.C.Q, and I.Y.R. As was the case for human GluR2, the relative frequency of the edited/unedited codons observed was also differentially regulated in an age specific manner. EAA4 cDNA clones isolated from human fetal brain (17-18 weeks gestation) revealed a relatively low editing efficiency. The majority of EAA4 cDNAs amplified from the cerebellum of a 2 year old female were of the hemi-edited I.C.R type. This variant was also the predominant type found in corpus striatum cDNAs. While not being bound by theory it is believed that in individuals of the same age, different editing states can exist in distinct neuronal populations. Thus the I.C.R form could have a role in the kainate receptor channel complexes in a specific neuronal tissue such as the cerebellum or corpus striatum while being rare in the brain as a whole. Hippocampus derived cDNAs revealed a distinctly different expression pattern of edited EAA4 with the majority of cDNAs examined being fully edited V.C.R. Editing in substantia nigra resulted in an approximately equal ratio of unedited I.Y.Q to fully edited V.C.R cDNA while no editing was observed in temporal cortex. Examination of Q/R editing frequency in EAA3 has also revealed a non-uniform distribution in human brain. Again fetal tissue harbours a higher ratio of unedited Q forms than is generally observed in adult brain. The cerebellar and temporal cortical tissues used in this study were isolated from the same 2 year old female and indicate a significant (p<0.05) reduction of EAA3 SUBSTITUTESHEET editing in the cerebellum. Hippocampal tissue isolated from another individual of the same age (female, 2 years) indicates editing at a similiar level to that of temporal cortex but again different to that of the cerebellum (p<0.05) . Editing efficiences were also found to be high for substantia nigra (60 years) and corpus striatum (57 and 63 years) . Having identified and confirmed a CNS receptor gene that is subject to editing, it is desirable to construct by application of genetic engineering techniques cells that produce forms of the receptor that are to be targetted in drug screening, e.g., one or more of the edited forms and/or the unedited form. According to one embodiment of the invention, the construction of such engineered cells, including both prokaryotic and eukaryotic cells, is achieved by introducing into a host cell a recombinant DNA construct in which DNA coding for a secretable form of the receptors, i.e. a form bearing its native signal peptide or a functional, heterologous equivalent thereof, is linked operably with expression controlling elements that are functional in the selected host to drive expression of the receptor-encoding DNA, and thus elaborate the desired receptor protein. Such cells are herein characterized as having the receptor-encoding DNA incorporated "expressibly" therein. The receptor-encoding DNA is referred to as "heterologous" with respect to the particular cellular host if such DNA is not naturally found in the particular host. The "unedited" receptor-encoding DNA can either be genomic in nature or, alternatively, can be derived from the protein sequence, i.e. cDNA. On the other hand, the "edited" receptor-encoding DNA can only be used in a cDNA form, as it does not exist in genomic form. The particular cell type selected to serve as host for production of a human receptor can be any of several cell types currently available in the art. It is, however, important that the cell type selected for production of receptor to be used in ligand screening assays will not cause editing of the receptor-encoding DNA to be expressed. According to one embodiment of the present SUBSTITUT invention, the cell line selected to serve as host for the production of a CNS receptor is a mammalian cell other than a human neuronal cell. Several types of such cell lines are currently available for genetic engineering work, and these include the Chinese hamster ovary (CHO) cells for example of Kl lineage (ATCC CCL 61) including the Pro5 variant (ATCC CRL 1281) ; the fibroblast-like cells derived from SV40-transformed African Green monkey kidney of the CV-l lineage (ATCC CCL 70) , of the COS-1 lineage (ATCC CRL 1650) and of the COS-7 lineage (ATCC CRL 1651) ; murine L-cells, murine 3T3 cells (ATCC CRL 1658) , murine C127 cells, human embryonic kidney cells of the 293 lineage (ATCC CRL 1573) , human carcinoma cells including those of the HeLa lineage (ATCC CCL 2) . A variety of gene expression systems have been adapted for use with these hosts and are now commercially available. Any one of these systems can be selected to drive expression of the human CNS receptor-encoding DNA. These systems, available typically in the form of plasmidic vectors, incorporate expression cassettes the functional components of which include DNA constituting expression controlling sequences, which are host- recognized and enable expression of the receptor-encoding DNA when linked 5' thereof. The systems further incorporate DNA sequences which terminate expression when linked 3' of the receptor-encoding region. Thus, for expression in the selected mammalian cell host, there is generated a recombinant DNA expression construct in which DNA coding for the receptor in secretable form is linked with expression controlling DNA sequences recognized by the host, and which include a region 5' of the receptor-encoding DNA to drive expression, and a 3' region to terminate expression. The plasmidic vector harbouring the recombinant DNA expression construct typically incorporates such other functional components as an origin of replication, usually virally-derived, to permit replication of the plasmid in the expression host and desirably also for plasmid amplification in a bacterial host, such as E.coli. To provide a marker enabling selection of stably transformed recombinant cells, the vector SUBSTITUTESHEET will also incorporate a gene conferring some survival advantage on the transformants, such as a gene coding for neomycin resistance in which case the transformants are plated in medium supplemented with neomycin. Included among the various recombinant DNA expression systems that can be used to achieve mammalian cell expression of the receptor-encoding DNA are those that exploit promoters of viruses that infect mammalian cells, such as the promoter from the cytomegalovirus (CMV) , the Rous sarcoma virus (RSV) , simian virus (SV40) , murine mammary tumor virus (MMTV) and others. Also useful to drive expression are promoters such as the LTR of retroviruses, insect cell promoters such as those regulated by temperature, and isolated from Drosophila, as well as mammalian gene promoters such as those regulated by heavy metals i.e. the metallothionein gene promoter, and other steroid-inducible promoters. The receptor-encoding DNA is incorporated for expression into any suitable expression vector, and host cells are transfected therewith using conventional procedures, such as DNA-mediated transformation, electroporation, or particle gun transformation. In this regard, it will be appreciated that the receptor-encoding DNA may be replaced with a synonymous codon equivalent of the isolated genomic sequence. Expression vectors may be selected to provide transformed cell lines that express the receptor- encoding DNA either transiently or in a stable manner. For transient expression, host cells are typically transformed with an expression vector harbouring an origin of replication functional in a mammalian cell. For stable expression, such replication origins are unnecessary, but the vectors will typically harbour a gene coding for a product that confers on the transformants a survival advantage, to enable their selection. Genes coding for such selectable markers include the E. coli gpt gene which confers resistance to mycophenolic acid, the neo gene from transposon Tn5 which confers resistance to the antibiotic G418 and to neomycin, the dhfr sequence from murine cells or E. SUBSTITUTESHEET coli which changes the phenotype of DHFR- cells into DHFR+ cells, and the tk gene of herpes simplex virus, which makes TK- cells phenotypically TK+ cells. Both transient expression and stable expression can provide transformed cell lines, and membrane preparations derived therefrom, for use in ligand screening assays. For use in ligand screening assays, cells transiently expressing the receptor-encoding DNA can be stored frozen for later use, but because the rapid rate of plasmid replication will lead ultimately to cell death, usually in a few days, the transformed cells should be used as soon as possible. Such assays may be performed either with intact cells, or with membrane preparations derived from such cells. The membrane preparations typically provide a more convenient substrate for the ligand binding experiments, and are therefore preferred as binding substrates. To prepare membrane preparations for screening purposes, i.e. ligand binding experiments, frozen intact cells are homogenized while in cold water suspension and a membrane pellet is collected after centrifugation. The pellet is then washed in cold water, and dialyzed to remove endogenous ligands, such as glutamate in the case of EAA receptors, that would otherwise compete for binding in the assays. The dialyzed membranes may then be used as such, or after storage in lyophilized form, in the ligand binding assays. Alternatively, intact, fresh cells harvested about two days after transient transfection, or after about the same period following fresh plating of stably transfected cells, can be used for ligand binding assays by the same methods as used for membrane preparations. When cells are used, the cells must be harvested by more gentle centrifugation so as not to damage them, and all washing must be done in a buffered medium, for example in phosphate-buffered saline, to avoid osmotic shock and rupture of the cells. The binding interaction between a ligand candidate and a human CNS receptor is evaluated typically using a predetermined amount of cell-derived membrane (measured for example by protein SUBSTITUTE SHEET determination) , generally from about 25μg to lOOμg. Generally, competitive binding assays will be useful to evaluate the affinity of a ligand candidate relative to an endogenous ligand, such as glutamate, serotonin or dopamine, depending on the type of receptor involved. This competitive binding assay can be performed by incubating the membrane preparation with radiolabelled endogenous ligand in the presence of unlabelled ligand candidate added at varying concentrations. Following incubation, either displaced or bound radiolabelled ligand can be recovered and measured, to determine the relative binding affinities of the ligand candidate and endogenous ligand for the particular receptor used as substrate. In this way, the affinities of various ligand candidates for the human CNS receptors of the present invention can be measured. As an alternative to using cells that express receptor-encoding DNA, ligand/receptor interaaction may also be determined electrophysiologically, e.g., using cells for example Xenopus oocytes, that yield functional membrane-bound receptor following introduction by injection either of receptor-encoding messenger RNA into the oocyte cytoplasm, or of receptor-encoding DNA into the oocyte nucleus. To generate the messenger RNA of cytoplasmic delivery, the receptor-encoding DNA is typically subcloned first into a plasmidic vector adjacent a suitable promoter region, such as the T3 or T7 bacteriophage promoters, to enable transcription into RNA message. RNA is then transcribed from the inserted gene in vitro, collected and then injected into Xenopus oocytes. Following the injection of nL volumes of an RNA solution, the oocytes are left to incubate for up to several days, and are then tested for the ability to respond to a ligand candidate supplied in a bathing solution. In the case of EAA receptors, which act in part by operating a membrane channel through which cations may selectively pass, the functioning of the receptor in response to a particular ligand candidate in the bathing solution may typically be measured as an electrical current utilizing microelectrodes inserted into the cell, in the established manner. SUBSTITUTESHEET Having expressed the edited and unedited forms of a receptor, it is desirable to distinguish the edited form of the receptor from its unedited counterpart. It is contemplated that certain neurodegenerative disease states may be associated with a dysfunctional editing mechanism. The functional differences between the edited and unedited forms of a receptor, thus, are valuable in screening compounds for potential therapeutic utility, e.g. to prevent over-activity of a particular function, such as channel activity, or to enhance a retarded receptor function. In this regard, a compound which is selective for either the edited or unedited form of the receptor is desirable. The edited and unedited receptor forms may be differentiated in terms of ligand binding characteristics, as described above, i.e. a ligand that binds to one form may be found to have little or no affinity for the other form. Thus, one method for determining selectivity of the edited and unedited forms of the receptor involves conducting comparative binding assays. Specifically, a cell encoding the edited form of the receptor is incubated with a test compound under appropriate conditions in the presence of an endogenous ligand, and the ligand binding affinity of that compound for that form of the receptor is determined relative to the endogenous ligand. This affinity is compared to the ligand binding affinity of the compound for the unedited form of the receptor determined in the same manner. Of course, the effects on receptor function of differential ligand binding characteristics should be considered in the instance that a compound exhibits a strong affinity for one receptor form while exhibiting a relatively weak affinity for the other receptor form. Alternatively, the edited and unedited forms of a receptor may be distinguished on the basis of electrophysiological function, particularly where EAA receptors are concerned. Electro- physiological function is determined by measuring the ligand- induced electrical current across a receptor-encoding cell, or a membrane preparation thereof, using a channel activity assay SUBSTITUTESHEET such as that described by Verdoorn et al. in Mol. Pharmacol. , 1988, 34:298. Briefly, the cell or membrane preparation is incubated in the presence of an endogenous ligand, for example glutamate, and the resulting electrical current is measured. It will be appreciated that the ligand preferentially bound by the receptor is the most suitable ligand with which to conduct these functional studies, e.g. kainate is the most suitable ligand for receptors that preferentially bind kainate, while AMPA is the most suitable ligand for receptors that preferentially bind AMPA. Differences in the electrophysiological function of the edited and unedited forms of the receptor can then be determined. As noted above, the effect of differentially binding test compounds on electrophysiological function can also be determined. With reference to the GluR2B receptor embodiment of the present invention, the functional differences between the unedited and edited forms of the receptor can be determined as described above. The ligand used to induce current flow in the GluR2B receptor is preferably AMPA. In the presence of AMPA, the unedited form of the receptor elicits an electrical current as it forms a ligand-gated ion channel which is permeable to divalent cations, and notably, permeable to calcium, while the edited form of the receptor does not elicit a current as it does form an ion channel which is permeable to divalent cations. In another embodiment of the present invention, DNA oligonucleotide probes are provided which facilitate the identification of genomic DNA encoding the unedited form of a protein, and to distinguish the cDNA version of edited mRNA from the cDNA version of unedited mRNA. The probes, comprising at least about 17 nucleotides, will correspond to the unedited region in the "unedited" genomic DNA, or to the edited region in the cDNA version of the "edited" mRNA sequence. As will be appreciated, a number of methods for using probes according to the present invention exist to successfully identify the target DNA sequence. In one method, for example, the probe is used as a hybridization probe in the usual manner. Thus, isolated SUBSTITUTESH ET immobilized DNA is combined with the probe under hybridization conditions, and the probe hybridizes to DNA having a corresponding sequence. Generally, in order to identify DNA/probe hybridization, the probe is labelled, e.g. by conjugation to a reporter molecule, such as a radiolabel, an enzyme label, a luminescent label or the like, using linker technology established for this purpose, or the probe incorporates in its structure a label such as a radioisotope of a molecule, e.g. 3 H and 13 C. To distinguish between the edited and unedited cDNA forms, high stringency conditions, and usually probes that are sequence complements of the target region, must be used due to the highly homologous nature of the two receptor forms. Another method for using probes according to the present invention is in the well-known PCR amplification procedure. In this method, a probe is prepared which incorporates the "unedited" codon at its 3' terminal end. The probe is incubated under PCR conditions with a genomic nucleic acid mixture, and if a sequence complementary to the probe is present, that sequence will be amplified. If, however, only a sequence encoding the "edited" version is present, the mismatched codon sequences will prevent PCR amplification from occurring. In another of its aspects, the invention provides an in vitro method for identifying agents that modulate the editing of human CNS receptors in vivo, which comprises: a) obtaining a human neuronal cell line that (1) incorporates DNA coding for the unedited form of an edited human CNS receptor, and (2) elaborates, upon culturing, the edited form of the receptor; b) culturing the cell line in the presence of a candidate modulator of said editing; and c) determining the effect of said modulator on the elaboration of said edited form of said receptor. SUBSTITUTE SHEET Particularly suitable as host cells for the construction of such cell lines are the human neuronal cell lines designated IMR-32 (ATCC CCL 127) , SK-N-MC (ATCC HTB 10) and SK-N-SH (ATCC HTB 11) . In one embodiment of the invention, the selected host is transformed to incorporate expressibly therein a polynucleotide that encodes the unedited form of the human GluR2B receptor, or the unedited form of the human EAA3 or human EAA4 receptor. Confirmation that the transformants express the edited form of the encoded receptor upon culturing can be obtained by constructing a cDNA library from message recovered from cells cultured under the conditions to be used during the assay, with editing being revealed by appropriate sequence alteration in cDNA encoding the chosen receptor target. With editing activity confirmed in the constructed host, the assay can then proceed simply by incubating the host in the presence of a chosen modulator of editing activity, and then again constructing a cDNA library from the RNA transcripts elaborated during culturing. Changes in the cDNA sequence at the predicted editing site reveal, correspondingly, an effect of the chosen modulator on the CNS receptor editing process. Specific embodiments of the present invention are described in the following specific examples which are not to be construed as limiting. Example l - Isolation of Genomic and cDNA encoding GluR2B The following PCR primers were used to amplify both genomic and cDNA sequences of GluR2B: PCR-1 5 'AACCTTGGCGAAATATCGCATCC 3 ' PCR-2 5 'GAGCCTCGGGATATCTATCATG 3 ' PCR-3 5 'ACACACCTCCAACAATGCGCCC 3 ' These primers were derived from coding regions of the cDNA sequence of GluR2B and are depicted in Figure 3. The PCR-1 and PCR-2 primers, which encode respectively a region of the DNA 5' to transmembrane domain I (TMI) and a region 3' of transmembrane SUBSTITUTESHEET domain II (TMII) , both of which are in a single exon, were used to amplify GluR2B genomic DNA (obtained from Clontech) . The PCR- 2 primer combined with the PCR-3 primer, which encodes a region in an adjacent codon, were used to amplify GluR2B cDNA (human ZAP cDNA libraries obtained from Stratagene) . The fact that the PCR- 3 primer corresponded to a region in a neighbouring exon ensured that only cDNA's were being examined and not contaminating fragments of genomic DNA (which would be much larger in size due to the presence of intron DNA between the two exons) . The DNA amplification reaction mixtures of both genomic and cDNAs contained: 100-500 ng DNA, 30 pmol of each primer, 5 units Taq polymerase (obtained from Promega) , 0.2 mM of each dNTP (in 10 mM KCl, 10 mM ammonium sulphate, 20 mM Tris-HCl, pH 8.8, 2 mM magnesium sulphate, 0.1% Triton). The conditions for the first 35 amplification cycles were as follows: 94 * C for 30 seconds, 55-61'C for 45 seconds and 72°C for 2 minutes. This was followed by a 10 minute incubation at 72°C. The amplified DNA was separated using gel electrophoresis, and the desired DNA fragments, i.e. a 294 bp fragment from the genomic DNA and a 326 bp fragment from the cDNA, were purified from the gel and subcloned into plasmid pT7blue (obtained from Novagen) for screening and sequencing. A comparison of the genomic and cDNA sequences identified a single nucleotide difference in the coding region of the transmembrane II domain at position 2134. Specifically, the genomic DNA included a G thereby encoding a glutamine, while the cDNA included an A thereby encoding an arginine. Example 2 - Frequency of RNA Editing of GluR2B The frequency of editing was determined using the plasmid DNA isolated according to Example 1. At the outset, the presence of the GluR2 insert (either genomic or cDNA) was confirmed by digestion with Bglll. Linearization of the plasmid DNA indicates TESH ET the presence of the GluR2 insert. The linearized plasmids were then tested for the presence or absence of editing. This was determined by Bbvl digestion, the recognition sequence for which is present in the unedited sequence. Thus, digestion of the unedited DNA with Bbvl yields two fragments, while digestion of the edited DNA with Bbvl yields a single fragment. The frequencies of edited vs. unedited GluR2B were as follows: Human GluR-2 Human GluR-4 TISSUE Q (CAG) R (CGG) * EDITED Q (CAG) R (CGG) * EDITED GENOMIC DNA 30 0 0 36 0 0 CORPUS STRIATUM 3 33 89 37 0 0 CEREBELLUM 0 37 100 35 0 0 FETAL BRAIN 2 51 96 33 0 0 HIPPOCAMPUS 0 43 100 38 0 0 SUBSTANTIA NIGRA 15 36 71 36 0 0 TEMPORAL CORTEX 0 43 100 28 0 0 Example 3 - Confirmation of RNA Editing of GluR2B Gene Initially, a Southern blot analysis was conducted in order to determine if two distinct GluR2B genes existed. Aliquots of human genomic DNA (8 μg) were individually digested with EcoRI, Hindlll, Pstl and Bglll restriction enzymes (obtained from New England Biolabs) . The digested DNA was then run on a 0.7% agarose gel, transferred to a nylon membrane and UV crosslinked. The immobilized DNA was hybridized to a purified TMI/TMII exon probe (i.e. the PCR-l/PCR-2 amplification product from Example 1) and radiolabelled with [o! 32 P]dCTP using the random priming method (Amersham) . Hybridization was carried out in 6x SSC (saline sodium citrate), 50% formamide, 5x Denhardt's solution, 0.5% SDS and 100 μg/ml sonicated salmon sperm DNA, at 42°C for 16 hours. The filters were washed with increasing stringencies up to 0.5x SSC/0.5% SDS, at 60°C for 20 minutes, before exposure SUBSTITUTESHEET to x-ray film at -80°C for 48 hours. The EcoRI, Hindlll and Pstl digests produced single bands upon hybridization with the TMI/TMII probe. This result was indicative of the existence of a single GluR2B gene which was known not to possess recognition sites in the TMI/TMII region for the EcoRI, Hindlll and Pstl enzymes. It was then necessary to determine if multiple exons were involved in the expression of the two forms of the GluR2B receptor. This was accomplished by digesting the genomic DNA with the Bglll restriction enzyme. The TMI/TMII exon includes a Bglll recognition site. Thus, a single exon would yield 2 bands, whereas multiple related exons would yield 3 or more bands. Only 2 bands of 5.5 kb and 2.2 kb were observed, confirming that the different forms of GluR2B are not as a result of multiple exons. The results of the analysis are illustrated in Figure 4. Example 4 - Expression of Unedited GluR2B Receptor For transient expression in mammalian cells, genomic and cDNA coding for the human GluR2B receptor is incorporated into the mammalian expression vector pcDNAl, which is available commercially from Invitrogen Corporation (San Diego, California, USA; catalogue number V490-20) . This is a multifunctional 4.2kb plasmid vector designed for DNA expression in eukaryotic systems. Incorporated on the vector are the CMV promoter and enhancer, splice segment and polyadenylation signal, an SV40 and Polyoma virus origin of replication, and M13 origin to rescue single strand DNA for sequencing and mutagenesis, Sp6 and T7 RNA promoters for the production of sense and anti-sense RNA transcripts and a Col El-like high copy plasmid origin. A polylinker is located appropriately downstream of the CMV promoter (and 3' of the T7 promoter) . To facilitate incorporation of GluR2B receptor-encoding cDNA into an expression vector, a Notl site is introduced onto the 5' flank SUBSTITUTESHEET of the Bluescript-SK cDNA insert, and the DNA insert is then released as a 3.4kb Hindlll/Notl fragment, which is then incorporated at the Hindlll/Notl sites in the pcDNAl polylinker. Sequencing across the junctions is performed to confirm proper insert orientation in pcDNAl. The resulting plasmid is then introduced for transient expression into a selected mammalian cell host, in this case cells of the COS-1 lineage (available from the American Type Culture Collection, Rockville, Maryland under ATCC CRL 1650) . The cells are transfected with approximately 8ug DNA per 10 6 COS cells by DEAE-mediated DNA transfection and treated with chloroquine according to the procedures described by Maniatis et al, supra. Briefly, COS-1 cells are plated at a density of 5 x 10 6 cells/dish and then grown for 24 hours in FBS-supplemented DMEM/F12 medium. Medium is then removed and the cells are washed in PBS and then in medium. There is then applied on the cells 10ml of a transfection solution containing DEAE dextran (0.4mg/ml), lOOuM chloroquine, 10% NuSerum, DNA (0.4mg/ml) in DMEM/F12 medium. After incubation for 3 hours at 37°C, cells are washed in PBS and medium as just described and are then shocked for l minute with 10% DMSO in DMEM/F12 medium. Cells are grown for 2-3 days in 10% FBS-supplemented medium, and at the end of the incubation, the dishes are placed on ice, washed with ice cold PBS and then removed by scraping. Cells are harvested by centrifugation at 1000 rpm for 10 minutes and the cellular pellet is frozen in liquid nitrogen, for subsequent use in ligand binding assays. In a like manner, stably transfected cell lines can also prepared using two different cell types as host: CHO Kl and CHO Pro5. To construct these cell lines, the DNA is incorporated into the mammalian expression vector pRC/CMV (Invitrogen) , which enables stable expression. The cDNA is inserted such that it is under the expression control of the cytomegalovirus promoter and upstream of the polyadenylation site and terminator of the bovine growth hormone gene, and into a vector background comprising the neomycin resistance gene (driven by the SV40 early promoter) as selectable marker. SUBSTITUTESHEET To introduce plasmids constructed as described above, the host CHO cells are first seeded at a density of 5 x 10 s in 10% FBS- supplemented MEM medium. After growth for 24 hours, fresh medium is added to the plates and three hours later, the cells are transfected using the calcium phosphate-DNA co-precipitation procedure (Maniatis et al, supra) . Briefly, 3μg of DNA is mixed and incubated with buffered calcium solution for 10 minutes at room temperature. An equal volume of buffered phosphate solution is added and the suspension is incubated for 15 minutes at room temperature. Next, the incubated suspension is applied to the cells for 4 hours, removed and the cells are shocked with medium containing 15% glycerol. Three minutes later, the cells are washed with medium and incubated for 24 hours under normal growth conditions. Cells resistant to neomycin are selected in 10% FBS- supplemented alpha-MEM medium containing G418 (lmg/ml) . Individual colonies of G418-resistant cells are isolated about 2-3 weeks later, clonally selected and then propogated for assay purposes. Example 5 - Ligand binding assay Transfected cells in the frozen state are resuspended in ice-cold distilled water using a hand homogenizer, sonicated for 5 seconds, and then centrifuged for 20 minutes at 50,000g. The supernatant is discarded and the membrane pellet stored frozen at -70°C. COS cell membrane pellets are suspended in ice cold 50mM Tris-HCl (pH 7.55, 5°C) and centrifuged again at 50,000g for 10 minutes in order to remove endogenous glutamate that would compete for binding. Pellets were resuspended in ice cold 50mM Tris-HCl (pH 7.55) buffer and the resultant membrane preparation is used as a tissue source for the binding experiments described below. Binding assays are performed using an amount of COS-derived membrane equivalent to from 25-100μg as judged by protein determination and a selected radiolabelled ligand. In SUBSTITUTESHEET particular, for AMPA-binding assays, incubation mixtures consisted of 25-100μg tissue protein and D,L-alpha- [5-methyl- 3 H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ( 3 H-AMPA, 27.6Ci/mmole, lOnM final) with 0.1M KSCN and 2.5mM CaCl 2 in the lml final volume. Non-specific binding is determined in the presence of lmM L-glutamate. Samples are incubated on ice for 60 minutes in plastic minivials, and bound and free ligand are separated by centrifugation for 30 minutes at 50,000g. Pellets are washed twice in 4ml of the cold incubation buffer, then 5ml of Beckman Ready-Protein Plus scintillation cocktail is added for counting. Example 6 - Isolation of Genomic and cDNA encoding GluR4 The procedure similar to that outlined in Example 1 was carried for another human CNS receptor, namely the GluR4 receptor, which is described in co-pending United States application serial no. 07/924,553. PCR primers for the same regions as those used for GluR2B were prepared and used to amplify GluR4 genomic DNA (obtained from Clontech) and cDNA (in human ZAP cDNA libraries obtained from Stratagene) . The isolated GluR4 genomic DNA and cDNA were compared for sequence discrepancies and none were found indicating that editing does not occur in the GluR4 human CNS receptor. Example 7 - Isolation of Genomic and cDNA encoding EAA3 and EAA4 The following PCR primers were used to amplify both genomic and cDNA sequences of EAA3: PCR5-2 5'-CTTGCAATCACAAAGAGTACACAG-3' PCR5-3 5' -CTGGTCGATAGAGCTTTGGG-3' PCR5-26 5'-CTCCAAACCCTTCATGACCC-3' 5int-1 5' - GGAAAATAGCAGGCTGGAATCGTATACCTTG-3' 5int-3 5' -CCCTACGAGTGGTATAACCCC-3' PCR6-1 5' -GCTGACCTTGCAGTTGCTCC-3' 8UB8TITUTE SHEET PCR6-2 5' -CCTGGCTATGACAAAGAGCAC-3' PCR6-3 5' -CTGGTGGACAGTGCTTTGGG-3' 6int-1 5' -GGCAAAATGGGCAACCGGTGTACCTTG-3' 6int-3 5' -CCACACCCTTGCAACCCTGA-3' Amplification - Using the mouse genomic structure and the human cDNA sequences, the above primers were designed to amplify both genomic and cDNA sequences of EAA3 and EAA4. The combinations PCR5-3/PCR5-26 and PCR6-1/PCR6-3 were used to amplify EAA3, EAA4, and EAA5 cDNAs respectively. These primers originate from separate exons thus ensuring only cDNAs were being examined and not potential genomic DNA contamination in the cDNA libraries. Genomic DNAs were examined by using the primer combinations 5- 2/5-26 and 5int-3/5int-l (EAA3) ; and PCR6-1/PCR6-2 and 6int- 3/6int-l(EAA4) . Human cDNAs were isolated from the bacteriophage lambda (λZAP) libraries of human cerebellum (female, 2 years) , hippocampus (female, 2 years) , temporal cortex (female, 2 years) , substantia nigra (male and female 60 years) , corpus striatum (caudate and putamen, males, 57 years) and fetal brain (female 17-18 weeks gestation) cDNAs (Stratagene Cloning Systems Inc., La Jolla, CA USA.; Cat.# 935201, 936205, 935205, 936210, 936213 and 936206 respectively) . DNA from these libraries was isolated essentially following the Qiagen Inc. (Chatsworth, CA USA) phage DNA preparation protocol. Human genomic DNA was obtained f om Clontech Laboratories Inc. (Palto Alto, CA USA) . The primer combinations were used to amplify EAA3 and EAA4 using either genomic or cDNA as a template, as previously described. PCR products of the correct sizes [PCR5-2/PCR5-26 (142bp) , PCR5- 3/PCR5-26 (315bp) , 5int-3/5int-l (138bp) , PCR6-1/PCR6-3 (474bp) , PCR6-1/PCR6-2 (221bp) and 6int-3/6int-l (127bp) ] were purified from an agarose gel and subcloned into pT7blue (Novagen Inc., Madison, WI USA) for screening and DNA sequencing. Southern blot analysis - 8 μg of human genomic DNA digested with single restriction enzymes (Hindlll, Pstl, BamHI and EcoRV) were electrophoresed on a 0.7% agarose gel, then transferred to a nylon membrane (Schleicher and Schuell Inc., Keene NH USA) . The DNA was immobilized on the membrane using UV radiation covalent SUBSTITUTESHEET crosslinking. Purified PCR6-1/PCR6-2 (EAA4, TMI) , 6int-3/6int-l (EAA4, TMII) and 5int-3/5int-l (EAA3, TMII) PCR amplification products were separately radiolabelled with [α- 32 P]dCTP by the random priming method (Amersham Corp. Arlington Heights IL USA) and used to probe the genomic DNA. Hybridizations were carried out in 6X standard saline citrate (IX SSC is 0.15M NaCl, 0.015M Na.citrate, pH 7.6), 50% formamide, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml sonicated salmon sperm DNA at 42°C for 16 hours. The filters were washed with increasing stringencies up to IX SSC/0.5%SDS, 60°C, for 20 min, before exposure to x-ray film at -80°C for 72 hours. Example 8 - Confirmation of RNA Editing of EAA3 and EAA4 genes RNA editing assay - Plasmid DNA was isolated and initially screened by restriction endonuclease digestion (Figure 8) . The occurrence of an internal restriction site [BstXI (EAA3) , EcoRV (EAA4) or BatπHI (EAA5)] was indicative of a correct sequence. The presence or absence of editing at the TMII Q/R site was determined by Bbvl digestion. BJvI has the recognition sequence 5 'GCAGC(N) β ... , 3 ' , and as such will cleave the unedited sequence (GCAGC) and leave intact the altered form (GCGGC) . A clear difference in the resulting restriction pattern of Q vs R forms provided a convenient method to categorize the clones. The TMI I/V and Y/C editing sites were confirmed by DNA sequencing, as were the smaller genomic fragments and a representative sample of TMII sites. SUBSTITUTE SHEET SOURCE OF CDNA EDITING CORPUS CEREBELLUM FETAL HIPPO¬ SUBSTANTIA TEMPORAL CORTEX STATUS STRIATUM BRAIN CAMPUS NIGRA EAA4 I.Y.Q 4 2 25 6 36 11 V.Y.Q 0 0 0 0 0 0 I.C.Q 0 0 0 0 0 0 V.C.Q 1 0 0 1 0 0 I.Y.R 0 0 3 0 1 0 V.Y.R 0 0 0 0 0 0 I.C.R 19 24 1 0 0 0 V.C.R 0 3 2 14 31 0 EAA3 (Q) 0 12 8 2 3 0 (R) 31 27 15 21 29 22 The table above reveals the relative frequency of TMI and TMII editing in EAA3 and EAA4 cDNAs amplified from different cDNA sources. The number of cDNA clones evaluated are listed according to their editing status and the tissue source. Example 9 - Isolation of Genomic and cDNA encoding EAA5 The procedure similar to that outlined in Example 7 was carried for another human CNS receptor, namely the EAA5 receptor, which is described in co-pending United States application serial no. 07/945,210, incorporated herein by reference. PCR primers for the same regions as those used for EAA4 were prepared and used to amplify EAA5 genomic DNA (obtained from Clontech) and cDNA (in human ZAP cDNA libraries obtained from Stratagene) . PCR7-2 5' -AATGATGCGTGTGGACAGGGC-3' , PCR7-3 5' -CCCCTGACCATCACCCATGT-3' , SUBSTITUTE SHEET PCR7-6 5' -CTGGCTCCGAGGTGGTGGAA-3' , PCR7-15 5' -CCTTTGGGCATCAGCACAGAC-3' , PCR7-16 5' -CTGGCGATGACGAAGAGGAC-3' , The isolated EAA5 genomic DNA and cDNA were compared for sequence discrepancies expected in the TMI/II regions, and none were found indicating that editing does not occur in the EAA5 human CNS receptor. However, further analysis revealed two variations of EAA5 cDNA which result in amino acid substitutions in the predicted extracellular amino-terminal region: Ser-310 ->Ala and Arg-352 -> Gin. These variations can be attributed to RNA editing involving T -> G and G -> A substitutions. SUBSTITUTESHEET

Claims: WE CLAIM : 1. A method for identifying a human CNS receptor ligand, which comprises : a) determining the interaction between a candidate ligand and a first human CNS receptor of a type that is subject to editing; b) determining the interaction between the candidate ligand and a second human CNS receptor which receptor is an editing- altered variant of said first receptor; and either c) selecting that candidate ligand which interacts selectively with one of said receptors, or d) selecting that candidate ligand which interacts substantially equivalently with both of said receptors. 2. A method according to claim 1, wherein said receptor is a human EAA receptor. 3. A method according to claim 2, wherein said EAA receptor is a human GluR2 receptor. 4. A method according to claim 3, wherein said EAA receptor is a human EAA3 receptor. 5. A method according to claim 2, wherein said EAA receptor is a human EAA4 receptor. 6. An isolated polynucleotide that codes for the unedited form of an edited human CNS receptor. 7. An isolated polynucleotide according to claim 6, wherein said receptor is a human EAA receptor. 8. An isolated polynucleotide according to claim 7, wherein said receptor is the unedited form of a human EAA receptor selected from a human GluR2 receptor, a human EAA3 receptor and a human EAA4 receptor. SUBSTITUTESHEET 9. An isolated polynucleotide that codes for a novel edited form of a human CNS receptor. 10. An isolated polynucleotide according to claim 9, wherein said receptor is a human EAA receptor. 11. An isolated polynucleotide according to claim 10, wherein said receptor is selected from a form of the human EAA4 receptor that incorporates at least one amino acid substitution selected from Val-532, Cys-536 and Arg-586. 12. A cell that incorporates expressibly therein a heterologous DNA molecule as defined in any of claims 6-11. 13. A method useful to identify agents that modulate the editing of human CNS receptors in vivo, which comprises: a) obtaining a human neuronal cell line that (1) incorporates DNA coding for the unedited form of an edited human CNS receptor, and that (2) elaborates, upon culturing, the edited form of the receptor; b) culturing the cell line in the presence of a candidate modulator of said editing; and c) determining the effect of said modulator on the elaboration of said edited form of said receptor. 14. A method according to claim 13, wherein said cell line incorporates DNA coding for the unedited form of a human EAA receptor. 15. A method according to claim 14, wherein said human EAA receptor is the human GluR2 receptor. 16. An edited form of the human EAA4 receptor, which incorporates at least one amino acid selected from Val-532, Cys- 536 and Arg-586. SUBSTITUTESHEET 17. An edited form of the human EAA4 receptor according to claim 16, which incorporates at least one of the amino acids selected from Val-532 and Cys-536. 18. An edited form of the human EAA4 receptor according to claim 16, which incorporates at least two of said amino acids. SUBSTITUTESHEET

Inventors: KAMBOJ RAJENDER, NUTT STEPHEN, KAMBOJ, RAJENDER, NUTT, STEPHEN
Applicants: ALLELIX BIOPHARMA, KAMBOJ RAJENDER, NUTT STEPHEN, ALLELIX BIOPHARMACEUTICALS INC., KAMBOJ, RAJENDER, NUTT, STEPHEN
Application year: 1994
Main classification: C12N15
IPC codes: C07K14, C12N5, C12N15, C12P21, C12R1, G01N33